Automated spectrophotometer apparatus and computer system for simulataneous measurement of a plurality of kinetic reactions

ABSTRACT

A spectrophotometer apparatus for the automatic positioning of multiple samples and sample blanks for measuring, for example, double differential absorbance, that is, sample absorbance with respect to both blank absorbance and time. The apparatus contains appropriate electronic hardware, for example suitable electronic computer and printout devices to generate a final digital printout of multiple analytical reaction rate results.

Unite States Patent 1191 MeCabe 1 1 Nov. 12, 1974 [5 AUTOMATEDSPECTROPHOTOMETER 3.628.682 12/1971 Paulson 356/246 APPARATUS ND COMSYSTEM 3,697,185 10/1972 Kassel et a1 356/205 3,748,044 7/1973 Liston356/184 FOR SIMULATANEOUS MEASUREMENT OF A PLURALITY OF KINETICREACTIONS OTHER PUBLICATIONS D. D. McCraken, A Guide To Fortran IVProgramming, John Wiley & Sons, New York, 1965.

Gilford Instrument Labs. General Catalog. Oberlin, Ohio (1968). p. 11.

Perkin; Elmer, Model 202 Catalog and Accessory Price List, (1968),Norwalk, Conn.

Gottschalk, Commissioner of patents, Benson et al.. 175 USPQ673 (1972).

Primary E.\aminerRonald L. Wibert Assistant Examiner-Paul K. Godwin [57]ABSTRACT 20 Claims, 8 Drawing Figures [76] Inventor: William C. McCabe,2335 W. 31st St., So, No. E, Wichita, Kans. 67217 [22] Filed: June 7,1972 [21] Appl. No.: 260,560

[52] 11.8. C1 356/205, 356/39, 356/96, 356/246 [51] Int. Cl. GOln 21/22[58] Field of Search 356/244, 246, 39, 96, 98, 356/205; 23/259; 233/26,11

[56] References Cited UNITED STATES PATENTS 2,169,601 8/1939 Corneliuset al 233/11 3,511,573 5/1970 lsreeli 356/244 3,531,211 9/1970 Staunton356/246 3,540,808 11/1970 Harmon et al 356/39 3,544,225 12/1970Wattenburg et al. 356/39 3,551,062 12/1970 Brown 356/244 3,567,3283/1971 Riley 1 356/96 3,589,814 6/1971 Patterson et a1 356/205 3,609,0479/1971 Marlow 356/205 MONO- CHROMATOR PHOTOVIETER PATENIEUIIUY 12 I974SIIEU 10F 4 PHOTOMETER IPEAK PICKER I (S-B) SIGNAL GENERATOR H T m E M 8WA VI m B n W 6 6 E E IVE m GR K flw m NE E Am E ,6 A R P I I w 6CHANNEL A F (LO G-DIGITAL CONVERTER) CONCENTRATION COMPUTER AMPLIFIERPATENTEUNUV 12 I974 3341485 sum 20? a PATENTEU 12 I974 3.847.486

SIEU 30? 4 PHOTCIVIE'ER MONO- CHROMATOR IIIIIIIIIIIIII PATENTEDHUY 121974 SHEET It (If 4 CHANNEL A (8-8) III (S- B) I CHANNEL B DIGITALPRINTER AMPLI FIER CONCENTRATION COMPUTER LOG-DIG! CONVERT MS-B) T IAMPLIFIER H PRINTERT AUTOMATED SPECTROPHOTOMETER APPARATUS AND COMPUTERSYSTEM FOR SIMULATANEOUS MEASUREMENT OF A PLURALITY OF KINETIC REACTIONSBACKGROUND OF THE INVENTION The present invention relates to an improvedmethod and apparatus for determining multiple analytical reaction rateresults, particularly for clinical enzyme reactions. More particularly,the present invention is directed to a method and apparatus for theproper positioning of a plurality of samples and sample blanks forsimultaneously making a plurality of rate measurements on each sampleusing a particular arrangement of electronic computer hardware toproduce a digital printout of the reaction rate results.

In the area of clinical chemical analysis, much work has been donetoward achieving completely automatic analytical capabilities. One ofthe areas of routine analytical measurement which has not yet obtained acompletely automated status with advantageous results is enzyme rateanalysis for heterogenous samples, such as for example, blood serum. Theinstrumentation presently available, such as for example, a centrifugetype analyzer, suffers from at least one of the following limitations.Existing apparatus for measuring clinical enzyme reactions do notperform analysis at a rate (samples per hour) sufficient for routinepatient screening using reactions specific for particular enzymeanalysis,

that is, many colorimetric procedures are utilized; (b) secondaryreactions are frequently present requiring sample blanks for correction;(0) existing systems are not capable of readily checking for linearityof reaction versus time; (d) the spectrophotometer error is related tothe extinction coefficient used in making calculations; and (e) noadequate means is provided for checking for inadequate substrates whilethe tests are being performed.

The elimination of the above sources of error requires the use of aprecise spectrophotometer system capable of making accurate measurementsin the ultraviolet region of the spectrum, the ability to include asample blank in all measurements, the ability to make simultaneousmeasurements on a large number of samples and blanks, the ability todetermine the linearity of the reactions and the ability to measure theblank corrected final absorbance and final blank absorbance.

There are three basic systems of positioning a plurality of samples orsamples and blanks in the light beam of a spectrophotometer or discretewavelength photometer. One of the systems is the flow-type systemwherein samples flow continuously through a flowsuffers from thefollowing limitations: It has a relatively slow rate of pumping fluidswhich limits the rate of sampling. Also, a relatively large volume ofsample is required together with a micro cuvette. Furthermore, whenusing such a system it is very difficult to make measurements. versustime. The start-stop flow system also suffers from many limitationsincluding a relatively slow rate of pumping fluids coupled with astart-stop requirement. Thus, the sample rate limiting factors are theflow rate and the length of time required for the stop phase of thecycle.

The second type of system for positioning a plurality of sample orsamples and blanks in the light beam of a spectrophotometer is a systemrequiring a linear backand-forth motion of discrete samples inindividual cuvettes. This system can also be either continuous or of thestart-stop type. An example of this type of system is the Gilford Model244 automatic cuvette positioncr. This type of device moves from cuvettenumber 1 to cuvette number 2 to etc., and then back to cuvette number 1,with a start-stop cycle. The limitations of this system are as follows:The space factor is a problem when considering the total length of aholder which would be required for testing 30 or more samples or'acombination of 30 samples and 30 blanks which would require 60 cuvettes.Also, the time needed to move the device from the last position to thefirst position would introduce a sampling rate limiting factor. Inaddition, multiple readings for each sample versus time would berequired. A further disadvantage is that the mechanism required forchanging the direction of the linear motion of the cuvette to give arelatively slow movement in one direction and a very fast reversemovement in the op posite direction, i.e., 30 to 60 times as fastdepending upon the number of samples and blanks being tested, tomaintain the same measuring sequence, would place sever mechanicalstrain on the system and complicate the entire apparatus.

The third type of device for positioning a plurality of samples orsamples and blanks in the light path of a spectrophotometer is thatinvolving the circular motion of the cuvettes containing the samples andblanks to be analyzed in a wheel assembly. This is the type of motionutilized in the apparatus of the present invention. The features of thistype of system are as follows. Optimum single measuring conditions areprovided wherein only a minimum sample volume is needed and whereinplanar cuvette windows or curved cuvette windows normal to the light atits point of incidence on the cuvette window are used to preventreflectance and refraction artifacts. The circular motion of cuvettes ina wheel assembly also enables optimum sample-blank differentialmeasurements to be made wherein a single optical system is utilized withonly a very small variable in time. Thus only a single monochromator(light source) and a single photometer (detector) is utilized for makingsample-blank differential measurements. The circular motion of thecuvette wheel assembly also provides a maximum rate of sequentialpositioning of the cuvettes in the light path of the spectrophotometer.There is no known linear motion device or flow system which can movecuvettes containing samples to be measured into and out of the lightpath as fast as the circular motion of the cuvette wheel assembly.Finally, the circular motion of the cuvette wheel assembly producesmaximum sample positioning in a minimum amount of space. Thus thecircular motion of the cuvettes in the wheel assembly produces optimumphysical and theoretical measuring conditions in a minimum amount oflaboratory space at a maximum rate of analysis.

SUMMARY OF THE INVENTION An object of the present invention is toprovide an improved method and apparatus for making automated kineticmeasurements for clinical enzyme reactions.

Another object of the present invention is to determine the linearity ofthe reaction by making a plurality of measurements on each sample.

Still another object of the present invention is to provide an improvedspectrophotometer apparatus for the automatic positioning of multiplesamples and sample blanks for measuring for example, double differentialabsorbance, that is, sample absorbance with respect to both blankabsorbance and time.

A further object of the present invention is to provide an improvedmethod and apparatus for measuring different types of reaction ratesduring the same analytical run.

A still further object of the present invention is to provide animproved method and apparatus for automatically substrating a blankmeasurement during an analytical run which is essential for clinicalenzyme analysis if rapid and error-free results are to be realized.

Still another object of the present invention is to provide an improvedmethod and apparatus for making multiple discrete measurements on aplurality of samples and sample blanks to affect simultaneous reactionrate analysis on all of the samples.

Another object of the present invention is to combine with the abovespectrophotometer apparatus, appropriate electronic hardware, includingelectronic computer and printout devices to generate a final digitalprintout of these simultaneous multiple kinetic measurements forclinical enzyme reactions.

Other objects and further scope of applicability of thepresent inventionwill become apparent from the detailed description given hereinafter; itshould be understood, however, that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

Pursuant to the present invention it has been found that theabove-mentioned disadvantages may be eliminated and a much improvedmethod and apparatus for making kinetic measurements for clinical enzymereactions may be obtained by utilizing a spectrophotometer takingadvantage of the circular motion of cuvettes containing samples andblanks in a wheel assembly and utilizing a single monochromator as alight source, a single photometer as a light sensitive detector and anelectronic system for generating a final digital printout of theclinical enzyme reaction rate results. According to the presentinvention, a cuvette wheel assembly adapted for circular motion isutilized. The purpose of the cuvette wheel assembly is to hold andsequentially position a number of cuvettes containing samples and blanksto be analyzed in the light path of a discrete wavelength or band-passspectrophotometer. The sequential positioning of the cuvettes in thelight path is obtained by rotating the circular cuvette wheel assemblyabout its axis utilizing appropriate motor and gear means. Thepositioning of the individual cuvettes in the light path must becontrolled to very close tolerance and this sequential positioning mustbe coordinated with the proper use of said motor-gear drive system aswell as the use of an electronic computer adapted to monitor theelectrical output of the photometer.

The general, overall operation of the apparatus ofthe present inventioncan be defined as follows. First of all, the cuvette compartments of thecuvette wheel assembly are brought to assay temperature by one of twothermostating systems which will be later defined. The individualcuvettes are then filled with the assay solutions and blank solutions atthe assay temperature and loaded into the cuvette compartments of thewheel. The cuvette position number one, which is the first sample, isthen moved to a position indicated by a reference line, said positionbeing at a point where the euvette is ready to pass through the lightpath of the monochromator. The wheel revolution indicator knob is thenturned to position 1. After a preincubation-lag time, which is sometimesnecessary, the start bottom is pushed to begin the measuring procedure.The pushing of the start button simultaneously starts the motor whichthrough a gear mechanism rotates both the euvette wheel and the idlergear assembly and activates the computer analytical system. Thus thereading and calculating cycle is started. The cuvette wheel first makesone complete rotation with no printout, storing the sample vs blankdifferential readings, hereinafter noted as (8-8). As the wheel startsits second rotation, the idler gear assembly automatically positions therevolution indicator switch and the computer and printer function toprint out the analytical result. On each additional or on each alternaterotation of the cuvette wheel, depending on idler gear assembly andcomputer data storage capabilities another set of data is printed out.This is continued until, for example, four sets of data (answers) foreach of a plurality of samples are generated. In the case where eachrotation has been set to take 30 seconds at 2 rpm. four sets of answerswill be generated in either 2 /2 minutes or 4 minutes. In the case wherethe cuvette wheel is designed to hold 30 samples, the correspondingrates of analysis will be either 720 samples per hour or 450 samples perhour. The pushing of the start button also activates a timer whichautomatically stops the rotation of the cuvette wheel after either thefifth revolution, which corresponds to 2 /2 minutes of operation orafter the eighth revolution, which corresponds to 4 minutes ofoperation. If linearity of reaction rate has not been achieved for allof the samples, the analysis can be readily repeated by merely pushingthe start button.

After an analysis has been completed, a check can be made for maskedelevated samples by switching from a concentration mode of operation toan absorbance mode of operation and then pushing the start button. Thisoperation generates a set of thirty sample vs blank differentialabsorbance readings in one rotation of the wheel, which takes 30seconds. If these readings do not match the corresponding sampleconcentration readings, then the samples should be repeated after firstdiluting them. In a similar manner, high levels of NADH (eoenzyme)oxidase can be checked by removing the sample cuvettes and replacingthem with their corresponding cuvettes. Then, with the mode switch setfor absorbance measurements, the start but ton is pushed. The cuvettewheel, by making one revolution, will generate a set of 30 sample-blankabsorbance readings. If any of these absorbance readings are well belowthe majority of the others, indicating a low concentration of NADHduring the assay, the amount of NADH added to these samples is doubledand their analysis is repeated. It is desirable to maintain an optimumNADH concentration during the time of analysis for all enzyme reactionsrequiring NADH for the analysis.

The above two checks coupled with the routine use of blanks and controlsas well as the use of multiple point kinetic method for analysis, asdefined by the present invention, substantially eliminates all of thecommon causes of gross error frequently found in all other presentlyused systems for clinical enzyme analysis. The system as defined by thepresent invention not only gives about a 5 to fold increase in the rateof analysis over most systems currently used for enzyme analysis butalso is one of the only systems presently available for semi-automatedenzyme analysis which produces results which are not subject to grossanalytical error.

The rate of rotation of the cuvette wheel is related to several factorsand must provide a balance between optical error and variation of sampleconcentration. As the r.p.m. is decreased, the ratio of optical error tototal signal decreases. Any optical error is due mainly to slightmisalignment, which decreases as the rate of rotation decreases. As ther.p.m. decreases, the absorbance for a given measurement increases whichproduces a greater signal. The limiting factors to consider whendecreasing the r.p.m. to minimize the measuring error are as follows. Indetermining the rate of sample analysis, three readings determine a lineand four readings allows for one of the readings to be disregarded, forexample the first or last reading. At one r.p.m., it takes either one ortwo minutes per result or four or eight minutes per assay, which isequivalent to four results. Many reactions are linear for only about twoto four minutes, therefore all readings after three minutes, forexample, are suspect. This supports two r.p.m. rather than one r.p.m.,thereby giving a maximum assay time of four minutes and at least 450samples analyzed per hour which is sufficient because a faster rate ofanalysis is not presently required.

One of the features of the present invention is to provide the necessaryinterfacing between the cuvette carrier wheel assembly and thespectrophotometer. This interfacing can be achieved by the use of apeakpicker mechanism. As each cuvette rotates into proper alignment inthe light path, the peak-picker means closes the circuit from thephotometer to the computer. Readings are always taken as pairs, that is,(83),, (8-8 etc.. Thus, a sample reading is taken and this reading isstored as the cuvette wheel moves and then a blank reading, that is, thesecond of the pair, is taken. Each pair of readings is used by thecomputer to generate a set of differential values. The (5-8)differentials for samples 1, 2, 3, etc., noted as (S-B),, (S-B) (S-B)etc. are either stored or used to caculate A(S-B) differentialsdepending on the idler gear assembly function. The ilder gear assemblycan be used to indicate to the computer which revolution the cuvettewheel is on, i.e., whether the wheel is on a (5-3) storage revolution oron a A(S-B) calculating revolution. The A(S-B) calculating revolution isalso the revolution on which a set of results is printed out.

There are at least three types of peak-picker means which can be usedfor proper interfacing, i.e., for closing the circuit from thephotometer to the computer when a cuvette rotates into proper alignmentin the light path of the monochromator. One possible way of using apeak-picker means for closing the circuit from the photometer to thecomputer is through the use of an interval timing device to close thecircuit at constant time intervals, A T. This type of device is notshown in the drawings. It would function as part of the computer means.A second peak-picker means is to use the idler gear means to indicatewhen a cuvette is in the proper position for taking transmittancereadings. For exam ple, the computer takes readings from the photometcrscontinuous output of electrical signals only when a micro-switch isclosed and it is closed only when the euvette is in proper alignmentwith the optical axis as indicated by a trip pin on the idler gearassembly. A third peak-picker means would be an electronic switch peakpicker. In operation, the light generated from the monochromator iscontinuous and the electrical output from the photometer is alsocontinuous with the change in signal from the photometer beingdetermined by variations in the amount of light reaching the photometer.The amount of light reaching the photometer, is determined by theposition of the cuvette carrier wheel relative to the optical axis(light path). When each cuvette is positioned with its optical faceperpendicular to this imaginary line (optical axis) the amount of lightreaching the photometer will be at a maximum. As the euvette movesthrough this position, the light reaching the photometer increases tothis maximum and then decreases. The decrease on either side of thismaximum is the result of light lost by reflection, defraction andincrease effective cuvette path length. This maximum signal can be usedto signal the computer to take a reading at this instant of time.

In a preferred embodiment of the present invention, an electronic switchcontained in the computer closes the circuit each time the detectorsignal reaches a maximum. In this embodiment after the first revolution,a trip-pin on the idler gear turns the revolution indicator to itsalternate position. This opens the (S-B), corresponds to the firstrevolution) circuit to channel A (see FIG. 3) and closes the (8-3)corresponds to the second revolution) circuit to the A(S-B) generator.This activates the sequential release of previously stored (S-B),signals from channel A to be used with each corresponding successive(83),, signal to generate the corresponding A(S-B) signal. The trip-pinalso activates a timer which turns the motor off after a time intervalsufficient for four revolutions of the cuvette wheel. Each (S-B) signalis stored in channel A and the corresponding A (S-B) signal is convertedto a digital concentration printout (answer). This operation continuesfor three additional revolutions, after which the motor is automaticallyshut off thereby stopping the analysis. Thus, four equivalent answershave been generated for each sample being analyzed. Thus, in thepreferred embodiment, the second, (S-B) reading is used with the first(S-B), reading (which has been stored) to determine a A (8-8) and thenthe second (S- B) reading is substituted for the first (S-B), reading inthe storage circuit. The difference between the above two signals orreadings, A( 8-8) is used by the computer to calculate a digital answer7 which is automatically printed out.

In an alternative embodiment of the electronic system utilized in thepresent invention, the wheel is rotated until an indicator light, notshown in the drawing, corresponding to the first of two coupledrevolution lights and the reference lines are matched. This is anindication that the double trip-pin on the idler gear has positioned therevolution indicator switch to close the (S-B) circuit to channel A. Themotor-on switch is then pushed which starts the rotation of the cuvettewheel thereby initiating the assaying of the samples. This step alsostarts an interval timer which automatically turns the motor off after atime interval suffieient for eight revolutions of the wheel. As eachcuvette rotates into proper alignment in the light path of themonochromator, a peak-picker means momentarily closes the circuit fromthe photometer to the computer. After the first revolution, the doubletrip-pin on the idler gear positions the revolution indicating switch toopen the (8-H), circuit and close the (S-B) circuit to channel B (seeFIG. 3a). At the same time, the sequential release of previously stored(S-B), signals from channel A is also activated to be used with eachcorresponding successive (S-B) signal to generate the corresponding A(8-8) signal. The A (S-B) signal is converted to a digital concentrationprintout (answer). After the second revolution, the double trip-pin onthe idler gear assembly positions the revolution indicator switch toopen the (-8) circuit and close the (S-B), circuit. The stored (S-B),signals are then used with the (S-B) signals as they are generated todetermine a A (S-B) signal. In this operation the storage circuit foreach sample is cleared and the entire operation is repeated to obtain asecond set of answers. The entire operation is repeated and the cyclecontinues for eight revolutions at which time the motor is automaticallyshut off, thereby stopping the analysis. Four equivalent answers havethus been generated for each sample being analyzed. As will be readilyrecognized, the only difference between the preferred and thealternative embodiments of the electronic system, as described above, isa factor of two in the rate of sample analysis.

In all probability, a combination of mechanical and electronic devicesdiscussed above will be necessary to realize the optimum interfacingbetween the cuvette holder wheel and the monochromator-photometersystem.

If it is technically difficult to obtain the proper combination usingcuvettes with planar windows, cuvettes with curved windows having acenter of curvature which is coincident with the axis of the cuvettecarrier wheel can be used. In this case, a slight uncertainty in theposition of the wheel at the time of measurement would not significantlyaffect the measurement. This would allow a simple timing device in thecomputer coupled with a synchronous motor-driven simple gear system tokeep the wheel and spectrophotometer in phase. There are two technicalproblems with this type of system. First of all, it is technically moredifficult to produce optically accurate curved optical glass cuvettewindows than it is to produce planar cuvette windows. Furthermore, mostspectrophotometers would not have the light focused properly to avoidreflectance and refraction losses at the curved surfaces of thecuvettes. A lens system could be utilized to eliminate this problem.

In positioning the cuvettes in the light path of the spectrophotometer,it is essential that each cuvette, at the time of measurement, ispositioned such that all light striking the optical surface (window) isnormal to said surface at the point of incidence. Two different meansmust be used depending upon whether planar or curved surfaces (windows)are utilized. ln the case of cuvettes with planar windows, a horizontalplane bi secting the wheel contains the optical axis which isperpendicular to the axis of rotation of the wheel. Each rectangularcuvette chamber is thus positioned in the wheel so that one of twoadjacent and mutually perpendicular sides of each chamber is parallel toa plane containing a radius of the wheel and the axis of rotation of thewheel. The cuvette can be automatically positioned in this chamberthrough the use of springs which force the cuvette against said twomutually perpendicular sides of the chamber. The light incident on thecuvette optical surface (window) is collimated (focused at infinity) andtherefore is normal to the cuvette surface at the point of incidence. Inthe case of cuvettes with curved windows, a horizontal plane bisectingthe wheel also contains the optical axis and is also perpendicular tothe axis of rotation of the wheel. The curved optical surfaces of eachcuvette are coincident with two series of vertically displaced circleswith fixed, but different radii for each series with all circles beingcentered around the axis of rotation of the wheel. The light incident onthe cuvette optical surface is focused on the axis of rotation of thewheel and therefore is normal to this surface at the point of incidence.The absolute rotational position of the wheel is not critical as long assome portion of the optical surface is in the light path at the timemeasurements are taken.

The fact that double differential" measurements, that is, the (S-B)differential and the differences in (8-8) differentials are being made,reduce considerably the optical requirements for matching the cuvettewith the spectrophotometer. The accuracy of any single transmittancereading is of only secondary importance because it is only thedifferential readings that are used in the analysis as defined by thepresent invention and any systemmatic errors, for example, opticalerrors, will cancel themselves out and thus not contribute in an adverseway to the final result. Because of this feature, plastic cuvettes couldbe readily produced through molding, rather than the more difficult toproduce optical glass cuvettes, thus eliminating the latter problem.Thus, either the planar cuvette window or the curved cuvette windowsystem could be used equally well as far as the final results areconcerned. From the above discussion it is apparent that the threesystems discussed above, that is, the motor-gear drive system, theplanar or curved cuvette system and the electronic computer system havesome flexibility with the final choice of systems being based upontechnical considerations.

As is well known in the spectrophotometry field of technology, it isimperative to maintain a constant environmental temperature for thecuvettes throughout the entire period of the reaction rate analysis. Inone of the features of the present invention, the cuvette wheel isprovided with a hollow axle-bearing system for the introduction andremoval of a thermostating liquid into and out of the cuvette wheel.Thus, the thermostatic liquid having a constant temperature controlledoutside of the spectrophotometer apparatus is introduced into andremoved from the cuvette compartment through the axis of the cuvettewheel. Incidentally. because of the manner in which the thermostatingliquid is circulated in the cuvette compartment around the cuvette, nothermostatic liquid is disposed between the monochromator and the samplecuvette as well as between the photometer and the sample cuvette.

In an alternative means for thermostating the cuvette carrier wheel, athermoelectric heating-cooling means could be used. For example, thewheel could be made of metal with thermoelectric heating-coolingelements provided therein. Electrical contacts could then be made withan exterior power supply through the hollow axle and bearing of thecuvette wheel. In such an arrangement, spring loaded carbon tip contactscould be mounted in the bearing with corresponding copper contact stripsmounted in the axle bearing seat.

To provide a substantially light-tight system from the monochromator tothe cuvette wheel and from the euvette wheel to the photometer atelescopic light pipe device has been found to be particularlyeffective. This feature provides a light-tight mechanical coupling ofthe stationary parts of the system, that is, the monochromator and thephotometer, with the constantly moving or rotating part of the system,that is, the euvette wheel. The light pipe device of the presentinvention can be telescopically adjusted between the monochromator andthe photometer to provide a substantially light-tight environmentbetween these elements and the rotating cuvette wheel. Alternatively, alarge black box can be utilized to enclose the entire system. One of theadvantages of using the box is that it could serve the dual function ofnot only providing a lighttight system when the wheel is in use butcould function as a storage box for the wheel when it is not in use. Thebox can be disposed upside down over the wheel assembly and fastenedpermanently to the wheel support rack. When the wheel is not in use thebox and wheel assembly could be removed and the box turned right sideup. The lid to the box, with a handle attached thereto, could then beattached thus making a convenient carrying and storage case for thewheel assembly.

It should be pointed out that the rate of revolution of the cuvettewheel is somewhat flexible as are the number of sample cuvettes in thewheel and the number of revolutions of the wheel required for a completeanalysis of all of the samples. The conditions specified above areessentially optimum for minimizing analytical error, including aconvenient number of samples for a single analysis and a rate ofanalysis which is sufficient to meet the needs of 'a primary user, thatis, a clinical chemistry laboratory. Because the reagent mixing step inthe analytical procedure is not part of the automated operation of thepresent invention, different types of reaction rate measurements can' bemade during the same analytical run. This is a particularly advantageousfeature of the present invention. The automated kinetic measuringspectrophotometer system of the present invention is the only such knowndevice which can automatically subtract a sample blank during ananalytical run. This is an essential step for clinical enzyme analysisif both rapid and error free results are to be realized.

DESCRIPTION OF THE DRAWINGS The present invention will become more fullyunderstood from the detailed description given hereinbelow and theaccompanying drawings which are given by way of illustration only andthus are not limitative of the present invention and wherein,

FIG. 1 shows a perspective view of the spectrophotometer apparatus ofthe present invention including a monochromator, a photometer, a cuvettewheel assembly, and an idler gear means;

FIG. la shows another embodiment of the idler gear means of the presentinvention;

FIG. 2 shows a plan view of the cuvette wheel in conjunction with atelescopic light pipe device, a monochromator and a photometer;

FIG. 2a shows a front view section of the individual cuvettecompartments in the cuvette wheel;

FIG. 2b shows a side view section of the individual cuvette compartmentsin the cuvette wheel;

FIG. 3 shows a schematic illustration of the computer elements startingfrom the point where the signals are taken from the photometer;

FIG. 3a shows a schematic illustration of another em bodiment of thecomputer elements utilized in the pres ent invention; and

FIG. 4 shows a schematic illustration of both a preferred embodiment andan alternative embodiment of the computer function of the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The spectrophotometer apparatusof the present invention according to FIGS. 1 and 1a comprises amonochromator 1 (light source) a photometer 2 (detector) and a cuvettewheel 3 provided with a motor-gear-axle assembly. The cuvette wheel ismounted for rotation about an axle assembly 4 between the monochromatorand the photometer. The axle assembly 4 is provided at its upper portionwith an axle drive gear 5. A synchronous motor 6, which is located inthe vicinity of the upper portion of the axle assembly is provided witha motor drive gear 7 which is adapted to engage the axle drive gear 5for rotating the cuvette wheel. The axle drive gear is also associatedwith an idler gear 8 of an idler gear synchronizing mechanism. Becausethe idler gear engages the axle drive gear, the idler gear remains insynchronous phase with the cuvette wheel at all times and can be usedtogether with other associated elements to trip switches indicating tothe electronic components the relative position of the wheel and whatelectronic function should be performed. The idler gear mechanism couldperform the following functions: It is adapted to indicate when acuvette is in the proper position for taking transmittance readings."For example, readings are taken from the photometers continuous outputof electrical signals only when a given micro-switch 9 is closed and itis closed only when the cuvette 10 is in proper alignment with theoptical axis, as indicated by a trip pin 11 disposed on the idler gearshaft 12 of the idler gear 8. Measurements are always taken as pairs,that is, (S-B) (S-B) etc. The (S-B) difference readings are thentransformed into A (S-B) readings using the computer methods and devicesdiscussed above and hereinafter. One electronic means capable ofhandling the above pairs of readings is embodied in the Gilford Model2430 oscillating cell (S-B) system. This device takes a sample reading,stores this reading, moves the cell holder linearly and takes a blankreading. Both readings are taken as voltage which is directlyproportional to absorbance. The difference between these measurements,i.e., (8-8) is determined and in this case recorded on a chart recorder.The (8-8) difference readings are then transformed into A (S-B) readingsas stated above, using the computer methods and devices discussed aboveand hereinafter.

The idler gear mechanism is provided at its upper end, that is, the endopposite the idler gear, with a bearing sleeve 13 which is adapted toreceive the idler gear shaft 12. The idler gear is constructed to beidentical in size with the axle drive gear of the axle assembly so thatthe rotation of the idler gear is synchronous with the rotation of thecuvette wheel. The synchronous motor, the axle for the cuvette wheel andthe idler gear mechanism is supported above the photometer by a supportrack 14. The upper portion of the support rack is provided with a motorsupport 15 for supporting the synchronous motor, a top-axle bearingsupport 16 for supporting the top portion of the axle for the cuvettewheel, an idler gear shaft bearing support 17 for supporting the idlergear mechanism and a revolution indicator support 18 for supporting therevolution indicator. The lower portion of the axle for the cuvettewheel is supported by a bottom axle bearing support 19 which is adaptedto be secured to the upper portion of the photometer. As one of thepossible arrangements of the support rack, right angle elements of thesupport rack are provided at their opposite ends with male and femaleend portions which provide for the ready assembly and disassembly of thesupport rack about the photometer.

In one of the advantageous features of the present invention, a hollowaxle 20 for the cuvette wheel is utilized, said hollow axle beingprovided with upper and lower axle bearing means provided with channels21 which communicate with conduit means 22 and 23 for introducing athermostating liquid into and removing it from the cuvette compartmentsof the cuvette wheel from a source outside of the spectrophotometer.Thus a thermostating liquid which is controlled to a desiredtemperature, depending upon the particular samples being analyzed isintroduced through the hollow axle of the cuvette wheel via conduitmeans 22 to the cuvette compartment which contains the individualcuvettes. The thermostating liquid is removed from the cuvettecompartment via conduit means 23 through the hollow axle for the cuvettewheel and returned to the point where the liquid is being controlled ata predetermined temperature outside of the spectrophotometer. Thecuvette wheel and the cuvette axle are provided with additional supportbrackets 24 which further stabilize the cuvette wheel axle with respectto the cuvette wheel. The individual cuvette compartments which containthe cuvettes are provided with a hinged cover to exclude extraneouslight from the cuvette compartments.

the reset mechanism for setting the switch in its proper firstrevolution position. The revolution indicating switch 25 is supported onsupport rack 14 by support frame 18.

FIG. 1a shows an alternative embodiment of the idler gear mechanism ofthe present invention wherein the sample-blank cuvette positionindicator (trip pin) 28 is provided with a number of pins 11corresponding to the number of sample-blank cuvettes disposed in theeuvette wheel. Because the sample-blank cuvette position indicator isattached to the idler gear shaft, it rotates with the rotation of thecuvette wheel. As the sample disposed in the cuvette wheel is rotatedinto light communication with the monochromator and photometer, theindicator 11 makes contact with a mechanical switch peak picker(micro-switch) 9 which enables the signal of the sample to be recordedat a time T,. The peak picker indicates to the computer that the signalof the next cuvette which contains a blank be recorded at time T Throughthe use of appropriate electronic components the difference (S-B) can bereadily computed and stored. The idler gear shaft is also provided witha double trip pin element 29 which is associated with an alternaterevolution indicator switch 30.

FIG. 2 shows a plan view of the cuvette wheel of the present inventionshowing the top of each of the euvettes 10 disposed in the cuvettecompartments 31 of the cuvette wheel. FIG. 2a. and 2b show a frontsectional view of the cuvette wheel at a plane parallel to the wheelaxis and a side sectional view of the cuvettes disposed in the cuvettecompartments, respectively. The individual cuvette compartments 32contain a hinged cover 33. The individual cuvettes are held in positionagainst the cuvette compartment walls by spring means 34 and 35. As canbe readily seen, the thermostating liquid 36 is free to travel throughthe cuvette compartments around the individual cuvettes. It should benoted that the cuvette compartments are provided with a front and backslit which is adapted to transmit the light from the monochromator tothe photometer. As can be readily seen, no thermostating liquid isdisposed between the monochromator and the sample or between thephotometer and the sample. This arrangement, of course, eliminates anyerror which might be introduced by a thermostating liquid interposedbetween the sample and these elements.

FIG. 2 also shows a plan view of the monochromatorphotometer-cuvettewheel wherein a telescopic light pipe 37 is disposed between themonochromator and the cuvette wheel as well as between the photometerand the cuvette wheel as a stray light trap. The telescopic light pipeprevents the light from the monochromator from being lost to theenvironment and furthermore it performs the same function with respectto the light received by the photometer. The telescopic light pipe alsoprevents light present in the environment from effecting samplemeasurements. Because of the telescopic feature, the monochromator andthe photometer can be moved toward or away from the cuvette wheel. FIG.2 further shows the use of felt pads 38 and a metal flange 39 to preventextraneous light from entering into the system. As can be readily seen,the euvette compartments are provided with slits on the inside and theoutside of the cuvette wheel adjacent to each cuvette sample.Advantageously, the slit should be smaller than the diameter of thelight pipe.

F IG. 3 shows a schematic illustration of the computer elements whichcan be utilized to produce a final answer from the appropriate signalgenerated by the photometer. The continuous electrical signal from thephotometer is fed into the peak-picker means of the computer. Thepeak-picker means picks the maximum signal from the photometer for eachof the samples being analyzed at time T I and then the maximum signal ofthe blank being analyzed at time T plus AT. The signals of the sample Sand the blank B are then fed into a signal generator which produces thedifferential reading (S-B). From this point the obtaining of a printoutof the answer can follow one of two courses. In the preferred embodimentof the present invention, during the first revolution, the revolutionindicator switch 25 is manually set to its data (S-B) storage mode,i.e., the circuit to channel A is closed and all (S-B) readings arestored in channel A. After the first revolution a trip-pin on the idlergear automatically turns the revolution indicator switch 25 to itsalternate position. This opens the (8-8), circuit to channel A, and atthe same time, the (5-8) circuit to the A(S-B) generator is closed. Thedifferential reading (S-B) represents that reading taken on the secondrevolution of the cuvette wheel. As the differential readings (53),, areintroduced into the A( 8-8) generator, the sequential release of thepreviously stored (S-B) signals are activated from channel A to beintroduced into the A(S-B) generator to be used with each correspondingsuccessive (S-B) signal to generate the corresponding A(S-B) signal. Atthe same time, a timer which turns the motor off after a time intervalsufficient for four revolutions of the wheel is activated. The (S-B)signal which was originally introduced into the A(S-B) generator is nowstored in channel A to be used in subsequent calculations and the A(S-B)signal is converted to a digital concentration printout (answer). Thisoperation continues for three additional revolutions, at which time themotor is automatically shut off stopping the analysis. Four equivalentanswers have thus been generated for each sample being analyzed.

FIG. 3a represents a schematic of the computer elements utilized in analternative embodiment for obtaining a digital printout of the answer.In this embodiment, the (S-B) differential reading is stored in channelA and noted as (S-B), differential readings. The differential readingsstored in channel A is the direct result of the closing of the (S-B),circuit to said channel as a result of the operation of the'double trippin on the idler gear. After the first revolution, the double trip pinon the idler gear positions the switch to open the (5-H), circuit andclose the (S-B) Circuit to channel B. At the same time it also activatesthe sequential release of previously stored (S-B), signals from channelA to be used with each corresponding successive (S-B) signal to generatea corresponding A(S-B) signal. The A(S-B) signal is then converted to adigital concentration printout (answer). The difference between theoperation of the electronic elements of FIG. 3a when compared to FIG. 3is that the differential reading (S-B) which is used to calculate aA(S-B) signal is not restored in channel A so that it can be used tocalculate a new A(S-B) with further readings. Rather, after the printoutof the A(S-B) answer, the (S-B) signal is removed from the computersystem so that in order to calculate a new A(S-B) signal for subsequentmeasurements, the (5-3), signals must be stored in channel A. Thus,eight revolutions of the cuvette wheel are necessary to generate fourequivalent answers for each sample being analyzed. This is to becompared with the preferred embodiment as shown in FIG. 3 wherein onlyfive revolu tions of the cuvette wheel are required to generate fourequivalent answers for each sample being analyzed.

FIG. 4 is a schematic drawing illustrating the computer functiondescribed with respect to FIGS. 3 and 3a. The signals received from thephotometer in the first revolution are fed into a computer to determinea set of (SB) differentials which are stored. The second set of (8-3)differentials generated on the second revolution of the wheel are usedalong with the said previously stored (S-B) differentials to generate aA (5-8) differential. In the preferred computer storage system thesecond set of (8-8) differentials, i.e., (S-B),,, remains stored to beused on the third revolution to generate a second set of A (8-8)differentials. This is the storage system illustrated schematically inFIG. 3. In the alternate storage system the second set of (5-8)differentials is not stored and the entire process must be repeated.This is the storage system illustrated schematically in FIG. 3a.Independent of which computer storage system is utilized, the A(S-B)signal is subsequently introduced into a log-digital converter, aconcentration computer, and an amplifier and finally a digital printoutof the answer is obtained.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be apparent to one skilled in the art areintended to be included within the scope of the follow ing claims.

I claim:

1. An apparatus for making automated kinetic measurements comprising astandard light source, a standard light sensitive detector and a cuvettewheel assembly disposed between said light source and light sensitivedetector, and mounted for rotation about an axle assembly, said cuvettewheel assembly containing a plurality of cuvettes containing the samplesto be analyzed, means for rotating the axle assembly, thereby providingthe sequential positioning of the samples in the light path of the lightsource, an idler gear assembly associated with the axle assembly and insynchronous phase with the cuvette wheel assembly, a peakpicker meansassociated with the detector indicating when a sample is in the properposition for recording transmittance readings received by the detectorand an electronic computer system associated with the peakpicker meansand the idler gear assembly for generating a final digital printout ofmultiple analytical reaction rate results.

2. The apparatus of claim 1, wherein the axle assembly is provided witha standard axle drive gear and the idler gear assembly is provided witha standard idler gear, said axle drive gear and said idle gearbeing-substantially the same size and being in engaging relationshipwith each other.

3. The apparatus of claim 1, wherein the peak-picker means is aninterval timing device which forms part of the electronic computersystem, said interval timing device functioning to close the circuitbetween the detector and the computer-system at constant time intervals,AT for recording signals received from the detector.

4. The apparatus of claim 2 wherein the peak-picker means comprises aplurality of trip-pins associated for rotation with the idler gearassembly, each of said trippins corresponding to a sample or blankcuvette and being adapted to sequentially engage a micro-switch whichmomentarily closes the circuit between the detector and the computersystem for recording signals received from the detector.

5. The apparatus of claim 1, wherein the peak-picker means is anelectronic switch which forms part of the electronic computer system,said electronic switch functioning to close the circuit between thedetector and the computer system when a maximum signal is received fromthe detector.

6. The apparatus of claim 1, wherein the computer system comprises asignal generator which generates differential readings for eachrevolution of the cuvette wheel, a storage channel for storing saiddifferential readings for the first of said revolutions, a doubledifferential generator for receiving the differential readings from saidstorage channel and said signal generator corresponding to the secondrevolution of the cuvette wheel, means for simultaneously releasing saidstored differential readings, means for converting the doubledifferential readings to a final digital printout of multiple analyticalreaction rate results and means for using the differential readingscorresponding to the second revolution of the cuvette wheel withsubsequent differential readings corresponding to a third revolution ofthe cuvette wheel to generate another digital printout, and means forcontinuing this process until the desired number of analyticalmeasurements have been made.

7. The apparatus of claim 6, wherein revolution indicator means areassociated with the idler gear assembly for indicating to the computersystem which differential readings are to be stored and whichdifferential readings are to be sent directly to the double differentialgenerator.

8. The apparatus of claim 2, wherein revolution indicator means areassociated with the idler gear assembly for indicating to the computersystem which differential readings are to be stored and whichdifferential readings are to be sent directly to the double differentialgenerator.

9. The apparatus of claim 7, wherein said revolution indicator meanscomprises a trip pin on the idler gear assembly operatively associatedwith a revolution indicator switch.

10. The apparatus of claim 1, wherein the computer system comprises asignal generator which generates differential readings for eachrevolution of the cuvette wheel, a storage channel for storing saiddifferential readings for the first of said revolutions, a doubledifferential generator for receiving the differential readings from saidstorage channel and said signal generator corresponding to the secondrevolution of the cuvette wheel, means for simultaneously releasing saidstored differential readings from said storage channel to said generatorthereby generating double differential readings, means for convertingthe double differential readings to a final digital printout of multipleanalytical reaction rate results and means for clearing the storagecircuit.

11. The apparatus of claim 10, wherein the means for clearing thestorage circuit comprises an alternate revolution indicator meansassociated with the idler gear assembly for indicating to the computersystem which differential readings are to be stored and whichdifferential readings are to be sent directly to the double differentialgenerator.

12. The apparatus of claim 1, wherein the cuvette wheel is provided witha hollow axle-bearing-conduit system for the introduction and removal ofsaid thermostating liquid into and out of a cuvette wheel.

13. The apparatus of claim 12, wherein thermostating fluid from astandard recirculating liquid thermostating means enters the hollowaxle-bearing-conduit system through a first conduit means axiallycommunicating with the bottom end of the hollow axle-bearing system forintroducing a thermostating liquid thereto, second conduit means providecommunication between said hollow axle-bearing and the hollow cuvettewheel assembly for introducing said thermostating liquid thereto, thirdconduit means provide communication between said hollow cuvette wheeland said hollow axle-bearing system for removing said thermostatingliquid from said cuvette wheel and fourth conduit means axiallycommunicating with the top end of the hollow axle-bearing system forremoving said thermostating liquid therefrom back to the said standardrecirculating liquid thermostating means.

14. The apparatus of claim 1, wherein said apparatus is utilized forstandard clinical enzyme reactions.

15. A method for making automated kinetic measurements comprisingplacing a plurality of samples (S) and blanks (B) to be analyzed in acuvette wheel, passing said samples and blanks in a circular pathsequentially through the light path of a spectrophotometer, measuringthe transmittance readings for each S vs B as they pass through saidlight path and generating a plurality of (S-B) differential readings forthe first revolution of the cuvette wheel, storing the (8-8)differential readings, generating a plurality of (8-H) differentialreadings for the second revolution of the cuvette wheel and generatingdouble differential readings A(S B) through the sequential release ofsaid previously stored (S-B) differential readings from said firstrevolution, converting the A(S-B) differential readings to a digitalconcentration printout of multiple analytical reaction rate results,using the (5-8) differential readings from the second revolution of thecuvette wheel with the (8-H) differential readings from the thirdrevolution of the cuvette wheel to generate new double differentialreadings and subsequent digital concentration printouts, and continuingthis procedure until the desired number of analytical measurements havebeen made.

16. The method of claim 15, wherein said method is utilized for standardclinical enzyme reactions.

17. The method of claim 15 wherein the signals for each of the samplesand blanks are recorded when the cuvette is rotated into properalignment with the optical axis of the spectrophotometer as indicated bythe momentary completion of an electrical circuit for each of saidsamples and blanks in synchronism with the alignment of said samples andblanks with the optical axis.

18. The method of claim 15, wherein the signals. received from thedetector for each of the samples and blanks are recorded at constanttime intervals, AT.

19. The method of claim 15, wherein the signals for each of the samplesand blanks are recorded when the cuvette is rotated into properalignment with the optical axis of the spectrophotometer as indicated bya maximum signal received from the detector.

20. A method for making automated kinetic measurements comprisingplacing a plurality of samples (S) and blanks (B) to be analyzed in acuvette wheel, pass ing said samples and blanks in a circular pathsequentially through the light path of a spectrophotometer, measuringthe transmittance readings for each S vs B as they pass through saidlight path and generating a plurality of (S-B) differential readings forthe first revolution of the cuvette wheel, storing the (S-B)differential readings, generating a plurality of (SB) differentialreadings for the second revolution of the cuvette wheel and generatingdouble differential readings A(S-B)- through the sequential release ofsaid previously stored (S-B) differential readings from said firstrevolution, converting the A(S-B) differential readings to a digitalconcentration printout of multiple analytical reaction

1. An apparatus for making automated kinetic measurements comprising astandard light source, a standard light sensitive detector and a cuvettewheel assembly disposed between said light source and light sensitivedetector, and mounted for rotation about an axle assembly, said cuvettewheel assembly containing a plurality of cuvettes containing the samplesto be analyzed, means for rotating the axle assembly, thereby providingthe sequential positioning of the samples in the light path of the lightsource, an idler gear assembly associated with the axle assembly and insynchronous phase with the cuvette wheel assembly, a ''''peak-picker''''means associated with the detector indicating when a sample is in theproper position for recording transmittance readings received by thedetector and an electronic computer system associated with thepeak-picker means and the idler gear assembly for generating a finaldigital printout of multiple analytical reaction rate results.
 2. Theapparatus of claim 1, wherein the axle assembly is provided with astandard axle drive gear and the idler gear assembly is provided with astandard idler gear, said axle drive gear and said idle gear beingsubstantially the same size and being in engaging relationship with eachother.
 3. The apparatus of claim 1, wherein the peak-picker means is aninterval timing device which forms part of the electronic computersystem, said interval timing device functioning to close the circuitbetween the detector and the computer-system at constant time intervals,Delta T for recording signals received from the detector.
 4. Theapparatus of claim 2 wherein the peak-picker means comprises a pluralityof trip-pins associated for rotation with the idler gear assembly, eachof said trip-pins corresponding to a sample or blank cuvette and beingadapted to sequentially engage a micro-switch which momentarily closesthe circuit between the detector and the computer system for recordingsignals received from the detector.
 5. The apparatus of claim 1, whereinthe peak-picker means is an electronic switch which forms part of theelectronic computer system, said electronic switch functioning to closethe circuit between the detector and the computer system when a maximumsignal is received from the detector.
 6. The apparatus of claim 1,wherein the computer system comprises a signal generator which generatesdifferential readings for each revolution of the cuvette wheel, astorage channel for storing said differential readings for the first ofsaid revolutions, a double differential generator for receiving thedifferential readings from said storage channel and said signalgenerator corresponding to The second revolution of the cuvette wheel,means for simultaneously releasing said stored differential readings,means for converting the double differential readings to a final digitalprintout of multiple analytical reaction rate results and means forusing the differential readings corresponding to the second revolutionof the cuvette wheel with subsequent differential readings correspondingto a third revolution of the cuvette wheel to generate another digitalprintout, and means for continuing this process until the desired numberof analytical measurements have been made.
 7. The apparatus of claim 6,wherein revolution indicator means are associated with the idler gearassembly for indicating to the computer system which differentialreadings are to be stored and which differential readings are to be sentdirectly to the double differential generator.
 8. The apparatus of claim2, wherein revolution indicator means are associated with the idler gearassembly for indicating to the computer system which differentialreadings are to be stored and which differential readings are to be sentdirectly to the double differential generator.
 9. The apparatus of claim7, wherein said revolution indicator means comprises a trip pin on theidler gear assembly operatively associated with a revolution indicatorswitch.
 10. The apparatus of claim 1, wherein the computer systemcomprises a signal generator which generates differential readings foreach revolution of the cuvette wheel, a storage channel for storing saiddifferential readings for the first of said revolutions, a doubledifferential generator for receiving the differential readings from saidstorage channel and said signal generator corresponding to the secondrevolution of the cuvette wheel, means for simultaneously releasing saidstored differential readings from said storage channel to said generatorthereby generating double differential readings, means for convertingthe double differential readings to a final digital printout of multipleanalytical reaction rate results and means for clearing the storagecircuit.
 11. The apparatus of claim 10, wherein the means for clearingthe storage circuit comprises an alternate revolution indicator meansassociated with the idler gear assembly for indicating to the computersystem which differential readings are to be stored and whichdifferential readings are to be sent directly to the double differentialgenerator.
 12. The apparatus of claim 1, wherein the cuvette wheel isprovided with a hollow axle-bearing-conduit system for the introductionand removal of said thermostating liquid into and out of a cuvettewheel.
 13. The apparatus of claim 12, wherein thermostating fluid from astandard recirculating liquid thermostating means enters the hollowaxle-bearing-conduit system through a first conduit means axiallycommunicating with the bottom end of the hollow axle-bearing system forintroducing a thermostating liquid thereto, second conduit means providecommunication between said hollow axle-bearing and the hollow cuvettewheel assembly for introducing said thermostating liquid thereto, thirdconduit means provide communication between said hollow cuvette wheeland said hollow axle-bearing system for removing said thermostatingliquid from said cuvette wheel and fourth conduit means axiallycommunicating with the top end of the hollow axle-bearing system forremoving said thermostating liquid therefrom back to the said standardrecirculating liquid thermostating means.
 14. The apparatus of claim 1,wherein said apparatus is utilized for standard clinical enzymereactions.
 15. A method for making automated kinetic measurementscomprising placing a plurality of samples (S) and blanks (B) to beanalyzed in a cuvette wheel, passing said samples and blanks in acircular path sequentially through the light path of aspectrophotometer, measuring the transmittance readings for each S vs Bas they pass through said light path and generating a plurality of (S-B)differential readings for the first revolution of the cuvette wheel,storing the (S-B) differential readings, generating a plurality of (S-B)differential readings for the second revolution of the cuvette wheel andgenerating double differential readings Delta (S-B) through thesequential release of said previously stored (S-B) differential readingsfrom said first revolution, converting the Delta (S-B) differentialreadings to a digital concentration printout of multiple analyticalreaction rate results, using the (S-B) differential readings from thesecond revolution of the cuvette wheel with the (S-B) differentialreadings from the third revolution of the cuvette wheel to generate newdouble differential readings and subsequent digital concentrationprintouts, and continuing this procedure until the desired number ofanalytical measurements have been made.
 16. The method of claim 15,wherein said method is utilized for standard clinical enzyme reactions.17. The method of claim 15 wherein the signals for each of the samplesand blanks are recorded when the cuvette is rotated into properalignment with the optical axis of the spectrophotometer as indicated bythe momentary completion of an electrical circuit for each of saidsamples and blanks in synchronism with the alignment of said samples andblanks with the optical axis.
 18. The method of claim 15, wherein thesignals received from the detector for each of the samples and blanksare recorded at constant time intervals, Delta T.
 19. The method ofclaim 15, wherein the signals for each of the samples and blanks arerecorded when the cuvette is rotated into proper alignment with theoptical axis of the spectrophotometer as indicated by a maximum signalreceived from the detector.
 20. A method for making automated kineticmeasurements comprising placing a plurality of samples (S) and blanks(B) to be analyzed in a cuvette wheel, passing said samples and blanksin a circular path sequentially through the light path of aspectrophotometer, measuring the transmittance readings for each S vs Bas they pass through said light path and generating a plurality of (S-B)differential readings for the first revolution of the cuvette wheel,storing the (S-B) differential readings, generating a plurality of (S-B)differential readings for the second revolution of the cuvette wheel andgenerating double differential readings Delta (S-B) through thesequential release of said previously stored (S-B) differential readingsfrom said first revolution, converting the Delta (S-B) differentialreadings to a digital concentration printout of multiple analyticalreaction rate results, using the (S-B) differential readings from thethird revolution of the cuvette wheel with the (S-B) differentialreadings from the fourth revolution of the cuvette wheel to generate asecond set of double differential readings and subsequent digitalconcentration printouts, and continuing this procedure until the desirednumber of analytical measurements have been made.